The present invention relates to the field of peptide syntheses and intermediates useful therein. More particularly, the invention relates to the synthesis of the polypeptide component of KL-4, a synthetic pulmonary surfactant.
The epithelium of mammalian lungs are lined with an endogenous pulmonary surfactant (PS) which facilitates breathing by aiding the transport of oxygen across the lung air-liquid interface. A deficiency in this surfactant is the primary cause of neonatal respiratory distress syndrome (RDS) and is linked to RDS in adults as well. Native PS is a mixture of lipids and proteins, and although its exact composition is unknown, researchers have prepared a number of exogenous surfactants which are useful in the treatment of RDS in pre-term infants. KL-4 is an example of an exogenous surfactant which is useful in the treatment of RDS as disclosed in U.S. Pat. Nos. 5,164,369, 5,260,273 and 5,407,914, hereby incorporated by reference.
KL-4 is a mixture of a pharmaceutically acceptable phospholipid and a 21 residue polypeptide, (L-lysine-(L-leucine)4)4L-lysine. (SEQ. ID No. 1) As disclosed in the aforementioned patents, this peptide was prepared by solid phase synthesis and recombinant DNA techniques. The solid phase synthesis comprises sequential addition of one or more amino acid residues coupled with suitable protection of amino or carboxyl groups. Although this process is effective, it is not amenable to the large scale synthesis necessary to manufacture a drug substance.
The object of the present invention is the production of the peptide component of KL4, namely (Lys-Leu4)4Lys (SEQ. ID No. 1) by a liquid phase peptide synthesis (xe2x80x9cLPPSxe2x80x9d). Unlike the methods disclosed in the aforementioned patents, this process is amenable to large scale synthesis.
An additional embodiment of the invention concerns the deprotection of the carboxy terminus of a peptide which was protected as an ester. Most peptide syntheses require manipulation of carboxyl and amino protecting groups. Typically, terminal carboxyl groups are protected as their ester derivative. However deprotection methods may be accompanied by racemization of the xcex1-carbon; a problem that is compounded as the length of the peptide increases. In several steps of the synthesis of (Lys-Leu4)4Lys, an ester protected carboxl group is deprotected. As with most biomimetic products, the configuration of the peptide is crucial and the active configuration of the residues of (Lys-Leu4)4Lys is xe2x80x9cLxe2x80x9d. This invention discloses a method of deprotecting a peptide""s ester protected carboxyl group which reduces the amount of racemized product. Although this method is applied in the synthesis of (Lys-Leu4)4Lys, it may be used in the synthesis of other peptides as illustrated hereinafter.
The invention relates to improved LPPS processes for the preparation of the 21 residue protein component of the pulmonary surfactant, KL-4, which is amenable to large scale synthesis. The first method uses three peptide fragments: 
The process starts off with a 3-residue right hand fragment which is successively reacted with the 5-residue body fragment to build an 18-amino acid fragment of the formula H-Leu-Leu-(Lys(Z)-Leu4)3-Lys(Z)-OBzl (SEQ. ID No. 3). This 18-amino acid fragment is then reacted with the 3-residue right hand fragment to form the final 21 residue protein of the present invention.
In the second method, a convergent synthesis is employed in which an 8-residue protected polypeptide of the formula: Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4) is prepared and saponified with tetraalkylammonium hydroxide. The saponified peptide is then reacted with a 13-residue peptide of the formula H-Leu-Leu-(Lys(Z)-Leu4)2-Lys-OBzl (SEQ. ID No. 5) to yield the protected 21-amino acid peptide. Removal of the protecting group by reaction with a suitable acid yields the final KL-4 polypeptide. This convergent method exhibits certain advantages in solubility and control over unwanted by-products, which makes the method particularly suitable to large scale synthesis.
In another aspect of the present invention is a novel method for the deprotection of the carboxy terminus of a peptide protected as an ester. This method comprises saponifying the protected peptide with tetraalkylammonium hydroxide reagent in a suitable solvent. This process provides a method of deprotecting the peptide which reduces the amount of racemization at the xcex1-carbon.
Herein, the amino acid nomenclature corresponds to standard conventions where: L-leucine is xe2x80x9cLxe2x80x9d or xe2x80x9cLeuxe2x80x9d, L-lysine is xe2x80x9cKxe2x80x9d or xe2x80x9cLysxe2x80x9d, L-alanine is xe2x80x9cAxe2x80x9d or xe2x80x9cAlaxe2x80x9d, benzyloxycarbonyl is xe2x80x9cZxe2x80x9d or CBZ, D-(1-naphthyl)alanine is xe2x80x9cD-Nalxe2x80x9d, 4-chlorophenylalanine is xe2x80x9cD-Calxe2x80x9d, L-serine is xe2x80x9cL-Serxe2x80x9d and D-3-pyridylalanine is xe2x80x9cD-Palxe2x80x9d. In addition all amino acid residue sequences are represented by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus. A dash at the beginning or end of the sequence indicates a bond to a radical such as H, OH or OBzl; and a dash in the middle of the sequence indicates a conventional amide bond. Other abbreviations and symbols are as follows: DMF is N,N-dimethylformamide, MeOH is methanol, HOBT is 1-hydroxybenzotriazole, THF is tetrahydrofuran, DCC is 1,3 dicyclohexylcarbodiimide, EtOH is ethanol, iPrOH is isopropanol, HBTU is N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-O-(1H-benzotriazol-1-yl)uronium-hexafluororphosphate), DIPEA is N,N-diisopropylethylamine, NMP is 1-methyl-2-pyrrolidone, HOOBT is 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, WSCD.I (water soluble carbodiimide) is 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and DIC is 1,3-diisopropylcarbodiimide. The term xe2x80x9calkylxe2x80x9d includes straight and branched groups; the term xe2x80x9chydroxidexe2x80x9d includes Group I metal hydroxides (NaOH, LiOH and the like) as well as tetraalkylammonium hydroxides; the term xe2x80x9csaltsxe2x80x9d includes tetraalkylammonium halides and tetrahalo borates and xe2x80x9cAcxe2x80x9d is acetyl.
The synthesis of (Lys-Leu4)4Lys (SEQ. ID No. 1) in accordance with the present invention, follows two pathways. Procedure A uses three peptides fragments: a 3-residue left-hand (amino-end), a three residue right hand (carboxy terminus) and a five residue body fragment to construct the molecule. 
As illustrated in Scheme A, H-Leu-Leu-Lys(Z)-OBzl and Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) are reacted together in the presence of a peptide coupling agent and an inert solvent at about 0xc2x0 C. to room temperature to give Boc-Leu-Leu-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-OBzl. (SEQ. ID No. 6) Examples of suitable peptide coupling agents include: DCC, DIC, HBTU, WSCDI, HOBT, HOOBt, where the preferred agents are HOOBT and HTBU. Solvents are chosen for their compatibility with the chosen coupling agent. Suitable solvents include DMF, THF, NMP and acetonitrile, where a mixture of DMF and acetonitrile is preferred. If the acid salt of H-Leu-Leu-Lys(Z)-OBzl is used, this peptide is neutralized with an organic base. The Boc protecting group is cleaved by treating Boc-Leu-Leu-Lys(Z)-Leu-Leu--Leu-Leu-Lys(Z)-OBzl (SEQ. ID No. 6) with an acid at about xe2x88x9220 to 0xc2x0 C. under an inert atmosphere. Although a solvent may be used with a gaseous acid, such as HCl, the preferred method uses neat trifluoroacetic acid at about 0xc2x0 C.
H-Leu-Leu-Lys(Z)-Leu4-Lys(Z)-OBzl (SEQ. ID No. 6) is treated with a peptide coupling agent, an organic base and Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) in an inert solvent at about xe2x88x924 to 10xc2x0 C. to give Boc-Leu-Leu-(Lys(Z)-Leu4)2-Lys(Z)-OBzl. (SEQ. ID No. 5) The preferred peptide coupling agents are HOOBT and HBTU, the preferred solvent is DMF and the preferred organic base is DIPEA. The Boc protecting group is cleaved by treating Boc-Leu-Leu-(Lys(Z)-Leu4)2-Lys(Z)-OBzl (SEQ. ID No. 5) with an acid at about xe2x88x9220 to 0xc2x0 C. under an inert atmosphere. The preferred acid was HCl and the inert solvent was ethyl acetate.
H-Leu-Leu-(Lys(Z)-Leu4)2-Lys(Z)-OBzl (SEQ. ID No. 5) is treated with a peptide coupling agent, an organic base and Boc-Leu-Leu-Lys(Z)-Leu-Leu-OH (SEQ. ID No. 2) in an inert solvent at about xe2x88x924 to 10xc2x0 C. to give Boc-Leu-Leu-(Lys(Z)-Leu4)3-Lys(Z)-OBzl. (SEQ. ID No. 3) The preferred peptide coupling agents are HOOBT and DIC, the preferred solvent is THF and the preferred organic base is DIPEA. The Boc protecting group is cleaved by treating Boc-Leu-Leu-(Lys(Z)-Leu4)3-Lys(Z)-OBzl (SEQ. ID No. 3) with an acid at about xe2x88x9220 to 0xc2x0 C. under an inert atmosphere.
H-Leu-Leu-(Lys(Z)-Leu4)3-Lys(Z)-OBzl (SEQ. ID No. 3) was treated with a salt, an organic base, a peptide coupling agent, water and Z-Lys(Z)-Leu-Leu-OH in an inert solvent at about 0 to 20xc2x0 C. over 2 to 5 h to give Z-(Lys(Z)-Leu4)4-Lys(Z)-OBzl. (SEQ. ID No. 1) The preferred coupling agents are DIC and HOOBT, the preferred solvent is THF and the preferred salt is LiBF4. The benzyl protecting groups are removed by hydrogenating Z-(Lys(Z)-Leu4)4-Lys(Z)-OBzl (SEQ. ID No. 1) in the presence of a catalyst and an inert solvent under a positive H2 atmosphere. The preferred catalyst is Pd/C, the preferred solvent is acetic acid and the preferred H2 pressure is 2 to 2.2 bar. 
Procedure B follows another approach. This method employs a convergent pathway where two larger fragments are combined to produce the protected final product. In addition this method uses tetrabutylammonium hydroxide to saponify an ester protected carboxy group with less than 1% racemization of the xcex1-carbon.
As illustrated by Scheme B, H-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 2) is treated with Boc-Lys(Z)-Leu-Leu-OH, a peptide coupling agent, an organic amine in an inert solvent at about xe2x88x925 to xe2x88x922xc2x0 C. for about 2 to 3 h to give Boc-Lys(Z)-Leu-Leu- Leu-Leu-Lys(Z)-Leu-Leu-OR. (SEQ. ID No. 4) The preferred coupling agents are HOOBT and HBTU, the preferred organic base is DIPEA, the preferred solvent is DMF and the preferred group for R is methyl. The terminal ester protecting group is saponified by treating Boc-Lys(Z)-Leu-Leu-Leu-Leu-Lys(Z)-Leu-Leu-OR (SEQ. ID No. 4) with a tetraalkylammonium hydroxide and water in an inert organic solvent at about xe2x88x9217 to 0xc2x0 C. over 10 to 160 min. Inert solvents include DMF and THF, tetraalkylammonium hydroxide reagents include tetrabutylammonium hydroxide, tetramethylammonium hydroxide, tetraethylammonium hydroxide and tetrabenzylammonium hydroxide. The preferred solvent is THF, the preferred tetraalkylammonium hydroxide is tetrabutylammonium hydroxide and the preferred temperature is xe2x88x925 to 0xc2x0 C. The reaction should be monitored by analytical methods, particularly HPLC in order to determine when the starting ester is saponified, for racemization increases over time.
Boc-(Lys(Z)-Leu4)Lys(Z)-Leu-Leu-OH (SEQ. ID No. 4) is treated with a salt, a peptide coupling agent, an organic base and H-Leu-Leu-(Lys(Z)Leu4)2-Lys-OBzl (SEQ. ID No. 5) in an inert organic solvent at about 0 to 25xc2x0 C. over 1 to 30 h to give Boc-(Lys(Z)-Leu4)4Lys(Z)-OBzl. (SEQ. ID No. 1) The preferred coupling agents are HOOBT and DIC, the preferred organic base is DIPEA, the preferred solvent is THF/water and the preferred salt is tetrabutylammonium chloride.
The protecting groups of Boc-(Lys(Z)-Leu4)4Lys(Z)-OBzl (SEQ. ID No. 1) are removed by treatment with trifluoroacetic acid followed by hydrogenation in the presence of a catalyst and an inert solvent under a positive H2 atmosphere. The preferred catalyst is Pd/C, the preferred solvent is acetic acid and the preferred H2 pressure is 2 to 2.2 bar. 
Although tetraalkylammonium hydroxides are employed in the synthesis of (Lys-Leu4)4Lys, (SEQ. ID No. 1) their utility is not restricted to the saponification of peptides with L-confirmations, nor to peptides of Lys or Leu. It is used in the saponification of Ac-D-Nal-D-p-Cal-OMe, where this peptide was treated with tetraalkylammonium hydroxide in an inert solvent at about xe2x88x9210 to 0xc2x0 C. to give Ac-D-Nal-D-p-Cal-OH. The preferred tetraalkylammonium hydroxide is tetrabutylammonium hydroxide and the preferred solvents are DMF and THF. This procedure gives 2.6% of the undesired L isomer while other methods, namely NaOH and aqueous acetone give 13% of the undesired diastereomer. In addition, tetraalkylammonium hydroxides are used in the saponification of Ac-D-Nal-D-p-Cal-D-3-Pal-L-Ser (OH)-OBzl. (SEQ. ID No. 7) The preferred solvent for this saponification is THF, the preferred hydroxide is tetrabutylammonium hydroxide and the preferred temperature is about xe2x88x926 to xe2x88x923xc2x0 C.
The following examples are meant to illustrate the invention, not to limit it. Other embodiments will be obvious to those skilled in the art and are claimed by this invention. The identity of the compounds was confirmed by HPLC and LC comparison with known standards. The purity of the compounds was determined by their HPLC area %, where the racemization % was determined by the same method.